Type of in vitro culture

Culture of intact plants (Seed orchid culture)Embryo culture (embryo rescue)Organ culture

1. shoot tip culture2. Root culture3. Leaf culture4. anther culture

Callus cultureCell suspension and single cell cultureProtoplast culture

Seed culture Increasing efficiency of germination of seeds

that are difficult to germinate in vivo

Precocious germination by application of plant growth regulators

Production of clean seedlings for explants or meristem culture

Embryo rescueThis is useful technique

in plant tissue culture where embryo abortion is reported at early stage and seed setting is failed.

Here immature embryo, ovary or ovule is rescued

( separated ) and cultured.

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Endosperm cultureAs endosperm is triploid so are the

plantlets formed from it.

This technique may be exploited as an alternative to crossing tetraploid and diploid parents for raising triploids during plant improvement programme.

The triploid plants are self-sterile and usually seedless.

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Organ culture Any plant organ can serve as an explant to

initiate cultures

Shoot apical meristem culture Culturing of apical meristem (the region of shoot apex

laying distal to leaf primordium)

Also known as meristem culture, meristemming & mericlones.

Extensively used in horticulture, agriculture and forestry.

Production of virus free germplasm

Mass production of desirable genotypes

Facilitation of exchange between locations (production of clean material)

Cryopreservation (cold storage) or in vitro conservation of germplasm

Root tip culture

By using meristematic cells such as those that operate in the root tip or bud.

Conceived by Kotte (germany) and Robbins (USA) in 1922.

leaf trace

procambium

tunicanew leaf

corpus

pithcortex

apical meristem

axillary meristem

Meristematic tissues ... Shoot ... apical,

… axillary

Meristematic tissues ... Shoot ... apical,

… axillary

LeafRoot

Meristematic tissues ... Shoot ... apical,

… axillary

LeafRootAdventitiousCambial tissues

Anther and pollen grain culture Haploid plants are derived from microspores

(pollen) cultured individually or in anthers

First report of Haploid plant from anther and pollen culture is by eminent Indian embryologists Guha & Maheshwari from the plant Datura stramonium.

Wheat, Rice, Maize, Rye, Tobacco, Potato, Brassicas has been used in pollen & anther culture to release the cultivars having superior chts. , high yield & disease resistance.

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Ovary or ovule culture Production of haploid plants

A common explant for the initiation of somatic embryogenic cultures

Overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers

In vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth

Bajaj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y. Yamada (eds.), Handbook of Plant Cell Culture. Volume 1. Techniques for Propagation and Breeding. MacMillan, New York. p. 228-287.

Callus Cultures Callus is formed via 3 developmental stages:1. Induction2. Cell division3. Differentiation

Callus formation is governed by The source of ExplantsNutritional composition of mediumAnd environmental factors

The unique feature of callus is that the abnormal growth has biological potential to develop normal root, shoot, and embryo ultimately forming plant.

In some instances it is necessary to go through a callus phase prior to regeneration via somatic embryogenesis or organogenesis

For generation of useful somaclonal variants

As a source of protoplasts and suspension cultures

For production of metabolites

Used in in vitro selection

Need for Callus Cultures

Cell suspension cultureThe technique of isolation and culturing a single

selected cell

When callus pieces are agitated in a liquid medium, they tend to break up.

Uses:Induction of somatic embryos and shootsIn vitro mutagenesis and selection of mutantsGenetic transformation studiesProduction of secondary metabolites etc.

Applications of plant cell culture

Suspension culture Suspension culture

Embryogenesis

Artificial seeds

Mutagenesis

Mutant

Protoplast

Cell modification

Cell fusionGene transfer

Secondary metabolites

Secondary products

Protoplast cultureCell without cell wall is called protoplast.For tissue culture application protoplast can be

isolated from almost any part of the plant but leaves are preferentially used for this purpose as these are easy to handle.Technique involves following steps;A. Sterilization of the explant by 2% sodium

hypochloride for about 30 minutes.B. Peeling of the epidermal cells.C. Digestion of cell wall by enzymes,

macerozymes, cellulase, hemicellulase and pectinase.

D. Washing of the released protoplasts.22

Protoplast fusionThe common methods employed for the fusion

of the released protoplasts is as:

A. Spontaneous fusion of protoplast.B. Mechanical induction fusion.C.NaNO3 induced fusion.D. High p H and Ca++ induced fusion.E. Poly Ethylene Glycol induced fusion (PEG).F. Electro fusion.

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The most popular method of protoplast fusion is PEG induced fusion.

In this freshly isolated protoplasts from desired parents are mixed in appropriate mixture.

The mixture is treated with 28-50% PEG for 15-30 minutes to facilitate fusion.

Protoplasts are subject to gradual wash with culture medium.

Fusion frequency can be increased by 50% if high Ca++ concentration is used at p H 9-10.

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Somatic embryogenesisThe process of a single cell or group of cells

initiating the development pathways that leads to reproducible regeneration of non zygotic embryo capable of germination to form complete plant.

OR a process embryo initiation and development

from somatic cell (non zygotic cell)

Haploid - Gametic number of chromosomes, n which may not be equivalent to x (x is one genomic complement)

Monoploid - haploid derived from a diploid, x (one genomic complement)

Polyhaploid - haploid from a polyploid (nx), prefix indicates genome complement number, e.g. tobacco is a dihaploid

Reduce time for variety development, e.g. 10 to 6 years or less

Homozygous recombinant line can be developed in one generation instead of after numerous backcross generations

Selection for recessive traits in recombinant lines is more efficient since these are not masked by the effects of dominant alleles

Agricultural applications for haploids - Rapid generation of homozygous genotypes after chromosome doubling

Processes Leading to Production of Haploid Plants

Androgenesis – haploid plant derived from male gamete, most common method in vitro

Parthenogenesis - from unfertilized egg

Apogamy - from other cells of the mega-gametophyte, example

Chromosome elimination - chromosome elimination in somatic cells, most common method used with plant breeding